Biomedical subjects

A Ide

Publications and source records attributed to A Ide.

At least 37 records · Page 2Linked to original sources

PubMed1996

In vitro synthesis of an N-myristoylated fusion protein that binds to the liposomal surface.

To increase the efficiency of association of tumor necrosis factor (TNF), a hydrophilic model protein, with liposomes, an N-myristoylation signal sequence was linked to the N-terminus of TNF by gene fusion. A DNA sequence coding for the N-myristoylation signal of Rasheed leukemia virus-gag protein was fused to be 5'-end of the cDNA coding for the mature domain of TNF to give N-myristoylated fusion TNF cDNA. In vitro translation of the mRNA coding for this fusion cDNA using rabbit reticulocyte lysate gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. Replacement of Gly2 in the myristoylation signal with Ala entirely inhibited the incorporation of [3H]-myristic acid into the fusion protein. A liposome binding assay using Ficoll density gradient centrifugation revealed that incubating the N-myristoylated fusion TNF with dipalmitoyl phosphatidylcholine-liposomes caused the complete binding of the protein to the liposomes, whereas much less of the nonmyristoylated counterpart bound. Thus, N-myristoylated fusion TNF, with high affinity for liposomes, was synthesized by the in vitro translation/transcription system.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of beta-N-acetylhexosaminidase from the liver of a prawn, Penaeus japonicus.

Beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS-PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS-PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The amino-terminal amino acid sequence was NH2-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-Val-?-Val-Lys-Gly- Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50 degrees C, respectively. The enzyme was stable from pH 4 to 11, and below 55 degrees C. It was 39% inhibited by 10 mM HgCl2. Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-beta-GlcNAcn, n = 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of Km and kcat at 25 degrees C and pH 5.5 were 0.137 mM and 598 s-1 for pNp-beta-GlcNAc, 0.117 mM and 298 s-1 for GlcNAc2, 0.055 mM and 96.4 s-1 for GlcNAc3, 0.044 mM and 30.1 s-1 for GlcNAc4, 0.045 mM and 14.7 s-1 for GlcNAc5, and 0.047 mM and 8.3 s-1 for GlcNAc6, respectively. These results suggest that this beta-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.

Amino Acid Sequence↗

PubMed1996

Inhibition of neutrophil-superoxide generation by alpha-tocopherol and coenzyme Q.

Effects of various derivatives of alpha-tocopherol (VE) and coenzyme Q (CoQ) on superoxide (O2.-) generation of neutrophils and protein kinase C (PKC) activity were examined. VE and CoQ8 inhibited O2.- generation of neutrophils stimulated by a protein kinase C mediated process monitored by cytochrome c reduction and spin trapping methods. The inhibitory action was observed not only with alpha-tocopherol, but also with beta-, gamma-, delta-tocopherols and with tocol which is a chemical similar to VE but lacking methyl groups on the chromanol ring structure and which is not a radical scavenger. By contrast, no inhibition was observed with 2-carboxy-2, 5, 7, 8-tetramethyl-6-chromanol (CTMC, trolox) or 2, 2, 5, 7, 8,-pentamethyl-6-chromanol (PMC) which are water soluble VE derivatives having radical scavenging activity. Compounds having a similar isoprenoid chain, such as CoQ, also have inhibitory activity on PKC-dependent O2.- generation of neutrophils. The inhibitory activity of CoQ derivatives is dependent on the length of the unsaturated isoprenoid chain. CoQ derivatives having 16, 24 and 32 carbon isoprenoid chains corresponding to CoQ4, 6, and 8 inhibited O2.- generation but 4 and 40 carbon isoprenoid chains corresponding to CoQ2 and 10 had no inhibitory activity on O2.- generation. Alpha-tocopherol and CoQ inhibited PKC activity but the ID50 for O2.- generation and PKC activity was different for each compound. However, no direct relationship between VE content and O2.- generation of neutrophils was observed. These results suggest that isoprenoids of VE and CoQ participate in the inhibition of the NADPH oxidase activation system through modulation of the neutrophil membrane probably by the inhibition of PKC.

Animals↗

PubMed1995

Treatment of cutaneous sarcoidosis with tranilast.

Tranilast is an anti-allergic drug clinically used for the treatment of atopy or urticaria. The drug has been shown to have an anti-fibrotic effect as well. We treated two cutaneous sarcoidosis patients with tranilast, resulting in remission within three months of administration. This drug should be an excellent tool for the treatment of other granulomatous diseases.

Adult↗

PubMed1995

Treatment of granuloma annulare with tranilast.

Three cases of granuloma annulare which did not exhibit a self-limited course were treated with tranilast at the dose of 300 mg/daily. The treatment resulted in the resolution of skin lesions within three months of administration. Although spontaneous resolution is often observed in granuloma annulare, tranilast may provide an alternative therapy for the treatment of cases resistant to spontaneous healing.

Adult↗

PubMed1995

Two patients with unusual skin lesions and circulating antikeratinocyte cell surface antibodies: detection of antibodies to the intracellular domain of the pemphigus foliaceus antigen (desmoglein) by studies using fusion proteins.

We report two patients with unusual skin lesions, who had antikeratinocyte cell surface IgG autoantibodies in their sera. Immunoblot analysis of both a human epidermal extract and a bovine desmosome preparation revealed that the sera of both patients reacted exclusively with the 160-kDa pemphigus foliaceus antigen (desmoglein). We further investigated the antigen molecule using bacterial fusion proteins produced by using bovine desmoglein cDNA, and found that both the sera reacted strongly and exclusively with the intracellular domain of the desmoglein. These results suggest that production of antidesmoglein autoantibodies in the present cases may be an epiphenomenon associated with damage to the keratinocyte cell membrane.

Aged↗

PubMed1995

Detection of autoantibodies against bullous pemphigoid and pemphigus antigens by an enzyme-linked immunosorbent assay using the bacterial recombinant proteins.

To develop a new method to evaluate autoantibodies in various autoimmune bullous skin diseases, we examined reactivity of bullous pemphigoid (BP) and pemphigus vulgaris (PV) patients' sera with partial bacterial fusion proteins of the 230 kD BP antigen (BPAG1) and PV antigen, respectively, by an enzyme-linked immunosorbent assay (ELISA), and compared the results with those of immunoblotting. We used two fusion proteins derived from the mouse BPAG1 and a fusion protein derived from the amino-terminus (EC1-2) of PV antigen. For both BP and PV sera, the ELISA scores were well correlated with the reactivities on immunoblot assays. The present study indicates that ELISA using recombinant antigen proteins in various autoimmune bullous skin diseases will be a new useful technique to detect the autoantibodies. However, development of recombinant proteins with the entire molecule and correct conformation will be necessary to establish a perfect ELISA system in the future.

Autoantibodies↗

PubMed1995

Human pro-tumor necrosis factor: molecular determinants of membrane translocation, sorting, and maturation.

Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane protein with a highly conserved 76-residue leader sequence. We have analyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplasmic, transmembrane, and linking domains. Cytoplasmic deletions included the Arg doublet at -49 and -48 and/or the Lys doublet at -58 and -57; additional mutants included deletion of residues -73 to -55 and -73 to -55, -49, and -48. The transmembrane and linking domain mutants included deletions in the -42 to -35 region, combined with the deletion of residues -32 to -1. Two hybrid mutants combined the cytoplasmic deletions with the deletion of residues -32 to -1. All of the cytoplasmic deletion mutants were properly translocated, as were the transmembrane deletion mutants with deletions up to residues -36, -35, -32 to -1, although the last one exhibited reduced efficiency; further incremental deletions, including deletions of residues -38 to -35 and -32 to -1, completely blocked translocation. Both hybrid mutants were effectively translocated; furthermore, transfection analysis revealed competent expression and maturation of both the cytoplasmic and hybrid mutants. Thus, proper expression and maturation of human pro-TNF can be accomplished with as few as approximately 12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.

Amino Acid Sequence↗

PubMed1995

The anatomical substrates for language and hemispheric specialization.

Three main lines of investigation are discussed in this paper: (1) the comparison between the anatomical arrangement of the language areas and the large-scale neurocognitive cortical networks partly involved in active or working memory; (2) the relations between hemispheric specialization and the development of interhemispheric communication; and (3) the analysis of individual differences in brain organization for language. The hypothesis and evidence presented stem from work being performed in our laboratories.

Cerebral Cortex↗

PubMed1994

Myristoylation of protein at a distinct position allows its phosphorylation by protein kinase C.

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid and three monomyristoylated lysozymes modified at a distinct position (at Lys-13, Lys-33, Lys-97) were isolated by two-step column chromatography. The relationship between membrane binding and phosphorylation by protein kinase C of these monomyristoylated lysozymes were examined using phospholipid vesicles. These three lysozymes bound to phospholipid vesicles to the same extent, whereas the binding of nonmyristoylated native lysozyme was negligible. When native and three monomyristoylated lysozymes were reacted with protein kinase C in a phosphatidylserine (PS)-containing vesicle system, phosphorylation was observed with the myristoylated lysozymes, whereas that of native lysozyme was negligible. However, a remarkable (more than sixfold) difference in the extent of phosphorylation by protein kinase C was observed among three monomyristoylated lysozymes with a different myristoylated position. These results suggest that the membrane binding of substrate protein is not sufficient for the phosphorylation by protein kinase C and the topology of the substrate protein on the membrane play a crucial role in the recognition of substrate protein by protein kinase C. These results further indicate that protein myristoylation can modulate the topology of the membrane-bound protein.

Amino Acid Sequence↗

PubMed1994

Bombesin enhances experimental carcinogenesis induced in rat colon by 1,2-dimethylhydrazine.

The effects of bombesin on the colonic mucosa and on the incidence, number, size and histology of colon cancers induced by 1,2-dimethylhydrazine (DMH) were studied in Fischer 344 rats. In experiment 1, rats were randomized into three groups to receive either saline or bombesin (10 or 30 micrograms/kg body wt) to determine the labeling index of normal colonic mucosa. In experiment 2, rats were given 20 weekly injections of DMH (20 micrograms/kg body wt) and received either saline or bombesin (10 or 30 micrograms/kg body wt) every other day for 24 weeks. Administration of bombesin significantly increased the labeling indices of colonic mucosa in a dose-dependent manner. Chronic administration of bombesin at both dosages with DMH caused significant increases in the incidence, number and depth of involvement of colon cancers; however, it did not affect the size and histological type of colon cancers. In addition, bombesin at the dose of 30 micrograms/kg significantly increased the labeling index of colon cancer. These results suggest that bombesin stimulates the cell proliferation of colonic mucosa and colon cancer and enhances colon carcinogenesis in rats.

1,2-Dimethylhydrazine↗

PubMed1994

Dysfunction of mouse liver mitochondria induced by 2,2'-azobis-(2-amidinopropane) dihydrochloride, a radical initiator, in vitro and in vivo.

Mouse liver mitochondria were uncoupled in a time dependent by intraperitoneal injection of a radical initiator, 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH) (100 mg/kg). State 3 respiration, ADP/O ratio and respiratory control ratio (RCR) were decreased 30 min after injection but there was no effect on state 4 respiration. Lipid peroxidation was increased and oxidative phosphorylation was uncoupled at one hr after drug injection but gradually recovered to normal levels after 14 hr in vivo. State 3 respiration, RCR and ADP/O ratio but not state 4 respiration of isolated mouse mitochondria were inhibited by short term incubation with AAPH in vitro. This inhibitory action was concentration dependent (ID50 = 5 mM) but was not prevented by alpha-tocopherol. AAPH had no effect on electron transport or the membrane potential of these isolated mitochondria. However, mitochondria were uncoupled via lipid peroxidation and swelling by long term incubation with AAPH. These inhibitory effects of AAPH were reduced by its spontaneous degradation not only in vitro but also in vivo. Thus AAPH induces mitochondrial dysfunction by direct action in the early period of treatment and free radicals produced from AAPH mediate mitochondrial swelling via lipid peroxidation in the late period. From these findings, it is concluded that mitochondrial phosphorylation plays an important role in the pathogenesis of liver injury induced by AAPH and that radicals generated by AAPH might be a source of liver injury and mitochondrial dysfunction in vivo.

Amidines↗

PubMed1992

Requirement of protein association with membranes for phosphorylation by protein kinase C.

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.

Adenosine Triphosphate↗

PubMed1992

Spontaneous remission with cyclic leukocytosis in chronic myelogenous leukemia.

A 41-year-old female with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia was observed for 7 years. Cytotoxic therapy was not instituted until blastic crisis developed in the last 4 months. Successive hematologic measurements revealed cyclic fluctuations in blood leukocyte and platelet counts. These cycles had an average duration of 71 days during the 43 months observed. The oscillation amplitude decreased gradually over time, followed by hematologic remission until the last 4 months. The possible relationship between long-term survival and cyclic leukocytosis is discussed.

Adult↗

PubMed1992

[An experimental study on portal infusion of 5-fluorouracil (the second report)].

This study was conducted to clarify the role of continuous portal infusion with 5-fluorouracil (FU) from two different standpoints. Experiment I (Assessment of the ability to reduce hepatic metastasis): Male Donryu rats (180-210 g) receiving intraportal inoculation of ascites hepatoma AH 60 C (4 x 10(6) cells) were divided into 3 groups; portal infusion group, venous infusion group and control. 5-FU (20 mg/kg/day) was continuously infused via either the portal vein or the femoral vein with heparin (100 U/kg/day) for 5 consecutive days. The portal infusion group alone showed a significant decrease in hepatic tumor volume on day 21 and prolonged survival time. Experiment II (Evaluation of the immunological effects): Animals without inoculation of AH 60 C were also randomized in the same way as Experiment I. There were no statistical differences among the 3 groups in total leukocyte counts, lymphocyte subpopulations or NK activities in the spleen, whereas hepatic NK activity was significantly decreased in the portal infusion group. Continuous portal infusion with 5-FU is considered effective to prevent lodging of tumor cells in the intra-hepatic portal system but might be disadvantageous for hepatic cellular immunity.

Animals↗

PubMed1991

Acute pancreatitis and Cushing's disease.

A 64-year-old woman with Cushing's disease died of acute necrotizing pancreatitis. This is the second case of this rare condition, in which acute necrotizing pancreatitis followed an ACTH-dependent hypercortisolism. The possibility that the tricyclic antidepressant drug, amitriptyline hydrochloride, induced the acute pancreatitis could not be ruled out; nevertheless, the present case may serve to add ACTH-dependent hypercortisolism to the possible causes of acute necrotizing pancreatitis.

Acute Disease↗

PubMed1989

Ricin A-chain induces fusion of small unilamellar vesicles at neutral pH.

The interaction of ricin and its constituent polypeptides, the A- and B-chain, with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC) was investigated by means of differential scanning calorimetry measurements. The A-chain, at neutral pH, entirely shifted the endothermic peak of small unilamellar vesicles of DPPC from 37 degrees C to 41 degrees C at low protein/lipid ratios. The potency of either ricin or the B-chain to induce the shift of endothermic peak was much less than that of the A-chain. The A-chain was also found to cause mixing of endothermic peaks of DMPC vesicles and DPPC vesicles. These data strongly suggest that the A-chain has the ability to induce fusion of phospholipid vesicles.

Calorimetry, Differential Scanning↗

PubMed1989

Studies on the optimal immunization schedule of the mouse as an experimental animal. The effect of antigen dose and adjuvant type.

This study was undertaken to establish the optimal immunogen dose for immunization of mice, using a viomycin-protein conjugate as a hapten immunogen. It was found that specific immunoglobulin G (IgG) formation depends on both the dose of antigen and the type of adjuvant: the optimal antigen dose for an immune response is quite different depending on whether the mice are being treated with Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FICA). The total IgG amount depends mainly upon the type of adjuvant used. FCA gave the double the level of IgG compared to that obtained with FICA. The antigen dose was found to have little influence on the total production of IgG. Mice given a primary immunization with 10 micrograms of antigen emulsified in FCA and then given a booster with the same amount of antigen emulsified in FICA produced a strikingly high level of specific anti-viomycin antibody of over 2.5 mg/ml of the antiserum. It was also found that decreases in the weight of the mice were related to the kind of adjuvant used as well as to the level of the specific antibody formed.

Adjuvants, Immunologic↗